1. Field of the Invention
This invention relates to novel methods for measuring cell growth rate in vivo and in vitro.
2. Background Information
Currently, there are two popular methods for measuring cell growth. One method is to count the number of cells at the beginning of an analysis period, and then count the number of cells at the cell of that period to measure the increase in cell number. Cell counting can be achieved by using microscopic methods with a hemocytometer or by instrument aided methods using a Coulter Counter or other flow cytometer. another Methodology for measuring cell growth is to determine the uptake of tritiated thymidine using beta counting methods. In this methodology, the cell number is determined at the initiation of the experiment and then tritiated thymidine is placed in with the cells. At periodic intervals, aliquots of the culture are removed, counted, and washed free of unbound tritiated thymidine. These washed aliquots are then subjected to Trichloroacetic Acid (TCA) precipitation followed by scintillation counting of the radioactively labelled solid precipitate to measure tritiated thymidine incorporation into DNA. This methodology merely measures the rate of DNA synthesis and does not measure the cell growth per se. Because of the relative ease of this methodology, however, it generally is the methodology of choice when looking at cell stimulation. Another method used to determine cell proliferation activity is to look at the number of mitoses per hundred cells in any tissue under examination. This methodology is not extremely accurate because the preparation procedure causes loss of cells. In general, these assays work well in vitro but are difficult to apply to measurements of cell growth in vivo.
Growth rate of tissues can be estimated by removing the tissue and monitoring in vitro pulse incorporation of tritiated thymidine. The tissue is sectioned into 30 micron sections and exposed to tritiated thymidine for thirty (30) minutes. The unincorporated tritiated thymidine is washed away and a nuclear emulsion is placed over the section where radioisotope disintegrations expose the film. The emulsion is developed and fixed; the tissue is stained with Hematoxylin and Eosin stain; then the section is examined microscopically to determine labelled fraction. This technique is labor intensive and time consuming.
Cyanine dyes have been used in various biological applications. Dioxacarbocyanine dyes have been used in performing white blood cell differential counts. Gunter Valet, Max Planck Ges Wissensch; Patent Accession Number 84-102307/17, Simultaneous Quantitative Determination of Blood Cells by Selective Staining and Measuring Volume and Fluorescence. The dyes utilized in these studies, however, are short chain carbocyanine dyes (less than ten carbons) and respond to changes in membrane potentials. Furthermore, the short chain carbocyanine dyes enter the cell's mitochondria, are cytotoxic, and, when the cells are washed, these dyes easily leak out of the cell whether or not the membrane potential of the cell is changed. Other short aliphatic chain cyanine dyes are used in many other biological assays. The short chain molecules, however, respond to membrane potentials and cross the cell membrane, penetrating into the mitochondria. H. M. Shapiro, U.S. Pat. No. 4,343,782, Aug. 10, 1982. The short chain dyes also are toxic to cells and cannot be used to determine cell growth rate.
Tricarbocyanine dyes (Fox, I. J., et al., Proc. May Clinic, 32: 478-484, 1957) and Evans-Blue dye (Schad, H., et al., Pfluegers Arch. Eur. J. Physiol., 370(2): 139-144, 1977) have been used in vivo to estimate cardiac output by a dilution method. Dow (Dow, P.,
Physiol. Rev., 36: 77-102, 1956) describes the method as injection of a known amount of some intravascular indicator on the venus side of the lungs, and measurement of the time course of arterial concentration of the indicator to determine the volume between the points of injection and sampling. These dyes are not used to stain cells.